The long-term objective of this proposal is to better understand the relationship between molecular structure and physiologic function in voltage-gated sodium channels (Navs). The specific aims focus on the role of two regions of cardiac Navs (hNavl.5), transmembrane segment 6 in domains 1 and 2 (D1-S6 and D2-S6), in an electrophysiologic property called slow inactivation. Navs are transmembrane proteins that play a critical role in the normal electrophysiology of excitable tissues, and proper function of Navs is dependent on specific structural characteristics. Amino acid mutations in Navs can alter normal physiologic function, and Nav mutations have been identified that underlie such human conditions as epilepsy, muscle myotonias, and sudden cardiac death syndrome. Several of these mutations affect slow inactivation and thereby alter membrane excitability and normal electrophysiologic function. This proposal addresses the hypothesis that conformational changes in D1-S6 and/or D2-S6 play an important role in the process of slow inactivation. To address this hypothesis, site-directed mutagenesis (alanine, lysine, or cysteine substitutions), a cellular expression system (HEK cells), and patch-clamp techniques will be used to study slow inactivation of wildtype and mutant hNav1.5. The SCAM (substituted-cysteine accessibility method) technique using methanethiosulfonate (MTS) agents and the cysteine-substituted mutants will be employed to look for molecular movement in these regions associated with slow inactivation. Experiments will be performed to compare slow inactivation in wild-type and mutants Navs. Cysteine-substituted mutants will be studied before and after MTS exposure using specific voltage protocols to assess state-dependent (e.g., slowinactivated) effects on MTS accessibility. The results will provide valuable information on molecular mechanisms and protein conformational changes during slow inactivation of Navs. This information will be useful for understanding heart Nav channelopathies such as long QT and Brugada syndromes.